| Research Abstract |
Calcium mobilization plays a central role in a variety of cellular responses such as insulin secretion by glucose in pancreatic β-cells. We have recently found that cyclic ADP-ribose (cADPR), a metabolite of NAD^+, is a novel second messenger for Ca^<2+> mobilization for insulin secretion. In the project, we have elucidated the molecular mechanism of cADPR signal transduction in mammalian cells.
1. We identified the binding sites for NAD^+ (substrate), cADPR (product) and ATP (regulator) for CD38 (mammalian ADP-ribosyl cyclase/cADPR hydrolase) by site-directed mutagenesis.
2. We isolated the human CD38 gene and determined its primary structure. The gene consists of 8 exons that extend -100 kbp on band p15 of chromosome 4 as a single copy gene. We found an Arg140→Trp mutation in Japanese NIDDM patients and the mutated protein exhibited a decrease in the enzymic activity of CD38 to form cADPR.
3. We found autoantibodies against CD38 that impair glucose-induced insulin secretion in Japanese
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and Caucasian subjects with diabetes.
4. We demonstrated that cADPR binds to FK506-binding protein 12.6 (FKBP12.6) on rat islet ryanodine receptor (RyR) and that the binding of cADPR to FKBP12.6 frees the RyR from FKBP12.6, releasing Ca^<2+>. In addition, in heart failure, the stoichiometry of FK506-binding protein per the ryanodine receptor was decreased. We isolated the human FKBP12.6 gene and determined the primary structure. The gene spans about 16 kbp on chromosome 2p21-23.
5. We found that the Ca^<2+> release from microsomes was greatly enhanced by the activation of CaM kinase II and A-kinase.
6. We produced knockout mice carrying a null mutation in the CD38 gene by homologous recombination and found that CD38 disruption impairs glucose-induced increases in cADPR, intracellular Ca^<2+> concentration, and insulin secretion.
7. Using CD38 knockout mice, we found that muscarinic Ca^<2+> signaling in pancreatic acinar cells involves a CD38-dependent pathway responsible for two cADPR-dependent Ca^<2+> release mechanisms in which the one sensitive to ryanodine plays a crucial role for the generation of repetitive Ca^<2+> spikes.
8. We determined the primary structure of streptococcal ADP-ribosyl cyclase/cADPR hydrolase. Using sitedirected mutagenesis, we found that Lys-162 and/or Lys-163 of the streptococcal enzyme, which correspond to Lys-129 of human CD38, participate in the cADPR binding and that Glu-307 of the streptococcal enzyme and Glu-226 of human CD38 are essential for the NAD^+ binding.
9. We found type 2 and type 3 ryanodine receptor Ca^<2+> channel (RyR) is expressed in normal pancreatic β-cells and the expression is markedly reduced in diabetic β-cells. We have made mutant mice lacking the both types of RyR gene in insulin producing β-cells. Less
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